The goal of this project is to determine the role of actomyosin structural dynamics in the mechanism of muscle contraction. Previous work on this project has demonstrated the power and complementary nature of EPR and optical spectroscopy in detecting the motions of myosin and actin in solution and in skinned muscle fibers. This work will continue, with increased emphasis on (1) the use of actin and myosin mutants to obtain new site-directed spectroscopic probe attachment sites and functional and structural perturbations, (2) the detection of structural dynamics under transient biochemical conditions, and (3) direct correlation of our spectroscopic measurements with structural analysis by x-ray crystallography and electron microscopy. The overall approach is to detect the structural dynamics of myosin and actin during their physiological interactions with each other and with ATP, designing experiments to test and refine specific molecular models for force generation. Aim 1 focuses on the development of improved spectroscopic methods for studying structural dynamics of myosin and actin in solution and in muscle fibers. In the remaining aims, these methods will be used to test specific hypotheses about the functional role of structural dynamics in the force-generating transition from weak to strong actomyosin binding: In Aim 2, probes are attached to myosin in solution, to test models for its structural dynamics during interaction with actin and nucleotides. In Aim 3, probes are attached to actin in solution, to test models for its structural dynamics during interaction with myosin-nucleotide complexes. In Aim 4, probes are attached to both actin and myosin in solution, to probe directly the structural dynamics of the actin-myosin interface. In Aim 5 probes are attached to myosin in skinned muscle fibers, to probe directly the coupling of structural dynamics to force generation. These spectroscopic studies will be coordinated with structural analysis by x-ray crystallography and cryo-EM, in order to provide a connection between 3D structure and the molecular dynamics of physiological function. The proposed research brings together a powerful combination of techniques, from molecular genetics to biochemistry to biophysics, to solve the molecular mechanism of muscle contraction. In particular, this work is of fundamental importance for understanding muscle function, and the technology generated by this work is already being used to provide molecular insight into muscle malfunction. More generally, this well-defined system serves as a model for studying the role of molecular dynamics and interactions in motor proteins, and the approaches we are developing should prove effective in the analysis of a wide range of problems in this field.